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World Congress on Biosensors 2014

World Congress on Biosensors 2014
Biosensors 2014

Friday, 28 September 2012

Just Published: Analytica Chimica Acta


A new issue of this journal has just been published. To see abstracts of the papers it contains (with links through to the full papers) click here:
Selected papers from the latest issue:

Preparation of molecularly imprinted polymers for organophosphates and their application to the recognition of organophosphorus compounds and phosphopeptides

28 September 2012, 08:37:53
Publication year: 2012
Source:Analytica Chimica Acta, Volume 748
Jun Haginaka, Hiromi Tabo, Hisami Matsunaga
Monodisperse molecularly imprinted polymers (MIPs) for diphenyl phosphate (DPP) and 1-naphthyl phosphate (1-NapP) have been prepared by a multi-step swelling and polymerization method using 4-vinylpyridine as a functional monomer, glycerol dimethacrylate as a crosslinker and cyclohexanol or 1-hexanol as a porogen. The retention and molecular-recognition properties of these MIPs for organophosphorus compounds were evaluated by HPLC using a mixture of phosphate buffer and acetonitrile as an eluent. In addition to shape recognition, hydrogen bonding and hydrophobic interactions could play an important role in the retention and molecular recognition of DPP and 1-NapP. Furthermore, the MIPs were applied to the separation of adenosine and adenosine phosphates (AMP, ADP and ATP). These phosphates were retained on the MIPs according to the number of phosphate groups in the molecule and were well separated from one another. Hydrogen bonding and hydrophobic interactions seemed to affect the retention and recognition of adenosine phosphates in low acetonitrile content, while hydrophilic interactions affected these properties in high acetonitrile content. Finally, the MIPs were applied to the trapping of phosphopeptides. The MIPs non-selectively trapped phosphopeptides, which have phosphorylated tyrosine, serine or threonine in the sequences, and successfully trapped four phosphopeptides in tryptic digests of bovine α-casein.

Graphical abstract

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Graphical abstract Highlights

► Monodisperse MIPs for organophosphates by multi-step swelling and polymerization. ► Application of MIPs to the separation of adenosine phosphates. ► Application of MIPs to the trapping of phosphopeptides in tryptic protein-digests.

Application of semi-permeable membrane dialysis/ion trap mass spectrometry technique to determine polybrominated diphenyl ethers and polychlorinated biphenyls in milk fat

28 September 2012, 08:37:53
Publication year: 2012
Source:Analytica Chimica Acta, Volume 748
Marek Roszko, Małgorzata Rzepkowska, Arkadiusz Szterk, Krystyna Szymczyk, Renata Jędrzejczak, Marcin Bryła
Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are hazardous food contaminants, their maximum legally allowable levels in food and environment are in the low pgg−1 range. Therefore some highly selective and sensitive analytical methods must be used to determine them. The 96/23/EC Directive implemented by EC Decision of 12 August 2002 requires recovery rate of an analyte at a concentration below 1ngg−1 within the 50–120% range at relative standard deviation (RSD) as low as possible. A method to determine low level PCBs and PBDEs in milk fat based on the semi-permeable membrane dialysis/ion trap GC MS technique was developed. Validation experiments proved that the method performance was within bounds set by the currently standing UE regulations. Recovery rates calculated on the basis of labeled internal standards for majority of the studied indicator PCB congeners and PBDE congeners were close to 100% at RSD below 20%. Also, dioxin-like PCBs recovery rates were compatible with the 1883/2006 EC Regulation (80–120%, RSD below 15%). The developed method turned out to be linear within a far broader concentration range than the studied 0.0025–10pgμL−1 range entirely sufficient for analyses of PCB and PBDE in milk fat. Within that range coefficient of linear correlation (R 2) of calibration curves exceeded 0.98.

Graphical abstract

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Graphical abstract Highlights

► We have investigated the usefulness of SPM dialysis for determination of PCB/PBDE. ► The factors affecting the dialysis process were evaluated. ► The ion trap MS was optimized for determination of PCB/PBDE in milk fat. ► Described extraction/clean up method proves usefulness for the milk fat analysis.

Determination of eight fluoroquinolones in groundwater samples with ultrasound-assisted ionic liquid dispersive liquid–liquid microextraction prior to high-performance liquid chromatography and fluorescence detection

28 September 2012, 08:37:53
Publication year: 2012
Source:Analytica Chimica Acta, Volume 748
M.M. Parrilla Vázquez, P. Parrilla Vázquez, M. Martínez Galera, M.D. Gil García
An ultrasound-assisted ionic liquid dispersive liquid–liquid microextraction (US-IL-DLLME) procedure was developed for the extraction of eight fluoroquinolones (marbofloxacin, norfloxacin, ciprofloxacin, lomefloxacin, danofloxacin, enrofloxacin, oxolinic acid and nalidixic acid) in groundwater, using high-performance liquid chromatography with fluorescence detection (HPLC-FD). The ultrasound-assisted process was applied to accelerate the formation of the fine cloudy solution using a small volume of disperser solvent (0.4mL of methanol), which increased the extraction efficiency and reduced the equilibrium time. For the DLLME procedure, the IL 1-octyl-3-methylimidazolium hexafluorophosphate ([C8MIM] [PF6]) and methanol (MeOH) were used as extraction and disperser solvent, respectively. By comparing [C8MIM] [PF6] with 1-hexyl-3-methylimidazolium hexafluorophosphate ([C6MIM] [PF6]) and 1-butyl-3-methylimidazolium hexafluorophosphate ([C4MIM] [PF6]) as extraction solvents, it was observed that when using [C8MIM] [PF6] the cloudy solution was formed more readily than when using [C6MIM] [PF6] or [C4MIM] [PF6]. The factors affecting the extraction efficiency, such as the type and volume of ionic liquid, type and volume of disperser solvent, cooling in ice-water, sonication time, centrifuging time, sample pH and ionic strength, were optimised. A slight increase in the recoveries of fluoroquinolones was observed when an ice-water bath extraction step was included in the analytical procedure (85–107%) compared to those obtained without this step (83–96%). Under the optimum conditions, linearity of the method was observed over the range 10–300ngL−1 with correlation coefficient >0.9981. The proposed method has been found to have excellent sensitivity with limit of detection between 0.8 and 13ngL−1 and precision with relative standard deviation values between 4.8 and 9.4% (RSD, n =5). Good enrichment factors (122–205) and recoveries (85–107%) were obtained for the extraction of the target analytes in groundwater samples. This simple and economic method has been successfully applied to analyse real groundwater samples with satisfactory results.

Graphical abstract

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Graphical abstract Highlights

► A novel method by US-IL-DLLME-LC-FD for fluoroquinolones determination. ► Simple, rapid and efficient method for water samples. ► Advantages over conventional methods. ► Low detection limits.

A new HPLC-DAD-MS/MS method for the simultaneous determination of major compounds in the crude extract of Lychnophora salicifolia Mart. (Brazilian arnicão) leaves: Application to chemical variability evaluation

28 September 2012, 08:37:53
Publication year: 2012
Source:Analytica Chimica Acta, Volume 748
Dayana Rubio Gouvea, Fernando Meloni, Arthur de Barros Bello Ribeiro, João Luis Callegari Lopes, Norberto Peporine Lopes
Lychnophora salicifolia Mart., which occurs in the Brazilian Cerrado in the states of Bahia and Minas Gerais as well as in the southeast of the state of Goiás, is the most widely distributed and also the most polymorphic species of the genus. This plant is popularly known to have anti-inflammatory and analgesic activities. In this work, we have studied the variation in terms of polar metabolites of ninety-three Lychnophora salicifolia Mart. specimens collected from different regions of the Brazilian Cerrado. Identification of the constituents of this mixture was carried out by analysis of the UV spectra and MS data after chromatographic separation. Twenty substances were identified, including chlorogenic acid derivatives, a flavonoid C-glucoside, and other sesquiterpenes. The analytical method was validated, and the reliability and credibility of the results was ensured for the purposes of this study. The concentration range required for analysis of content variability within the analyzed group of specimens was covered with appropriate values of limits of detection and quantitation, as well as satisfactory precision and recovery. A quantitative variability was observed among specimens collected from the same location, but on average they were similar from a chemical viewpoint. In relation to the study involving specimens from different locations, there were both qualitative and quantitative differences among plants collected from different regions of Brazil. Statistical analysis revealed that there is a correlation between geographical localization and polar metabolites profile for specimens collected from different locations. This is evidence that the pattern of metabolites concentration depends on the geographical distribution of the specimens.

Graphical abstract

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Graphical abstract Highlights

Statistical analysis revealed that there is a correlation between geographical localization of Lychnophora salicifolia samples and polar metabolites profile for specimens collected from different locations.
► A new HPLC-DAD-MS method for analysis of the crude extract of L. salicifolia leaves. ► Twenty substances were identified. ► Ninety-three wild specimens were analyzed. ► A correlation between geographic isolation and decrease of the chemical similarity. ► The pattern of metabolites depends on the geographical distribution of the specimens.

Characterizing uranium oxide reference particles for isotopic abundances and uranium mass by single particle isotope dilution mass spectrometry

28 September 2012, 08:37:53
Publication year: 2012
Source:Analytica Chimica Acta, Volume 748
M. Kraiem, S. Richter, N. Erdmann, H. Kühn, M. Hedberg, Y. Aregbe
Uranium and plutonium particulate test materials are becoming increasingly important as the reliability of measurement results has to be demonstrated to regulatory bodies responsible for maintaining effective nuclear safeguards. In order to address this issue, the Institute for Reference Materials and Measurements (IRMM) in collaboration with the Institute for Transuranium Elements (ITU) has initiated a study to investigate the feasibility of preparing and characterizing a uranium particle reference material for nuclear safeguards, which is finally certified for isotopic abundances and for the uranium mass per particle. Such control particles are specifically required to evaluate responses of instruments based on mass spectrometric detection (e.g. SIMS, TIMS, LA-ICPMS) and to help ensuring the reliability and comparability of measurement results worldwide. In this paper, a methodology is described which allows quantifying the uranium mass in single micron particles by isotope dilution thermal ionization mass spectrometry (ID-TIMS). This methodology is characterized by substantial improvements recently achieved at IRMM in terms of sensitivity and measurement accuracy in the field of uranium particle analysis by TIMS. The use of monodisperse uranium oxide particles prepared using an aerosol generation technique developed at ITU, which is capable of producing particles of well-characterized size and isotopic composition was exploited. The evidence of a straightforward correlation between the particle volume and the mass of uranium was demonstrated in this study. Experimental results have shown that the uranium mass per particle can be measured via the ID-TIMS method to a relative expanded uncertainty of about 10% (coverage factor k =2). The availability of reliable and validated methods for the characterization of uranium particles is considered to be essential for the establishment of SI-traceable measurement results. It is therefore expected that the method developed in this study is valuable for the certification of particulate materials in which the isotopic composition and the content of uranium must be accurately known.

Graphical abstract

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Graphical abstract Highlights

► A method to quantify the U mass in single micron particles by ID-TIMS was developed. ► Well-characterized monodisperse U-oxide particles produced by an aerosol generator were used. ► A linear correlation between the mass of U and the volume of particle(s) was found. ► The method developed is suitable for determining the amount of U in a particulate reference material.

A mass spectrometric method to determine activities of enzymes involved in polyamine catabolism

28 September 2012, 08:37:53
Publication year: 2012
Source:Analytica Chimica Acta, Volume 748
Shunsuke Moriya, Kaori Iwasaki, Keijiro Samejima, Koichi Takao, Kohfuku Kohda, Kyoko Hiramatsu, Masao Kawakita
An analytical method for the determination of three polyamines (putrescine, spermidine, and spermine) and five acetylpolyamines [N1-acetylspermidine (N1AcSpd), N8-acetylspermidine (N8AcSpd), N1-acetylspermine, N1,N8-diacetylspermidine, and N1,N12-diacetylspermine] involved in the polyamine catabolic pathway has been developed using a hybrid tandem mass spectrometer. Heptafluorobutyryl (HFB) derivatives of these compounds and respective internal standards labeled with stable isotopes were analyzed simultaneously by TOF MS, based on peak areas appearing at appropriate m/z values. The isomers, N1AcSpd and N8AcSpd were determined from their fragment ions, the acetylamidopropyl and acetylamidobutyl groups, respectively, using MS/MS with 13C2-N1AcSpd and 13C2-N8AcSpd which have the 13C2-acetyl group as an internal standard. The TOF MS method was successfully applied to measure the activity of enzymes involved in polyamine catabolic pathways, namely N1-acetylpolyamine oxidase (APAO), spermine oxidase (SMO), and spermidine/spermine N1-acetyltransferase (SSAT). The following natural substrates and products labeled with stable isotopes considering the application to biological samples were identified; for APAO, [4,9,12-15N3]-N1-acetylspermine and [1,4,8-15N3]spermidine (15N3-Spd), respectively; for SMO, [1,4,8,12-15N4]spermine and 15N3-Spd, respectively; and for SSAT, 15N3-Spd and [1,4,8-15N3]-N1-acetylspermidine, respectively.

Graphical abstract

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Graphical abstract Highlights

► Compounds in polyamine catabolic pathway were determined by a column-free ESI-TOF MS. ► N1- and N8-acetylspermidine were determined by a column-free ESI-MS/MS. ► The method was applied to determine activities of APAO, SMO, and SSAT in the pathway. ► The assay method contained stable isotope-labeled natural substrates. ► It is applicable to biological samples containing natural substrate and product.


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Thursday, 27 September 2012

Just Published: Journal of Chromatography B


A new issue of this journal has just been published. To see abstracts of the papers it contains (with links through to the full papers) click here:
Selected papers from the latest issue:

Liquid chromatography–tandem mass spectrometry method for simultaneous determination of seven commonly used anticancer drugs in human plasma

27 September 2012, 09:32:50
Publication year: 2012
Source:Journal of Chromatography B, Volume 906
Jingya Zhou, Shouhong Gao, Feng Zhang, Bo Jiang, Qin Zhan, Fei Cai, Jingxian Li, Wansheng Chen
This paper describes the development and validation of a novel, general liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the simultaneous determination of cyclophosphamide, ifosfamide, irinotecan, etoposide, gemcitabine, carboplatin and pemetrexed concentrations in human plasma. Samples were prepared by two kinds of extraction method and analyzed using a gradient separation over an Atlantis T3-C18 column (2.1mm×100mm, 3μm, Waters). Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of acetonitrile–water (0.1% formic acid and 10mM ammonium acetate) at a flow rate of 0.25mL/min. Linear coefficients of correlation were >0.992 for all analytes. The intra- and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 9.2%, while the accuracy was within ±10.5%. The mean recovery of all the analytes ranged from 50.0 to 81.0%. This method was successfully applied to clinical samples from cancer patients.

Highlights

► We developed a novel, sensitive, reproducible, and accurate LC–MS/MS method. ► This method could simultaneous determination of seven anticancer drugs in human plasma. ► This new method was successfully applied to clinical samples from cancer patients.

Multi-detection of preservatives in cheeses by liquid chromatography–tandem mass spectrometry

27 September 2012, 09:32:50
Publication year: 2012
Source:Journal of Chromatography B, Volume 906
Fabio Fuselli, Chiara Guarino, Alessandro La Mantia, Lucia Longo, Angelo Faberi, Rosa Maria Marianella
The incorrect use of preservatives in cheeses may compromise food safety and damage consumers. According to the law, more than one preservative may be contemporarily used in cheeses. So a method for their contemporary detection may be useful for both manufacturers and control agencies quality control. In this research a liquid chromatography–tandem mass spectrometric with electrospray ionization method for the multi-determination of seven preservatives (benzoic acid, citric acid, hexamethylenetetramine, lysozyme, natamycin, nisin and sorbic acid) in cheese was developed. The preservatives were contemporarily extracted from cheese by a single procedure, and analyzed by RP-LC/ESI-MS/MS (Ion Trap) in positive ionization mode, with single reaction monitoring (SRM) acquisition. Three sample types (hard, pasta filata and fresh cheese) were used for method evaluation. Recoveries were mostly higher than 90%; MDLs ranged from 0.02 to 0.26mgkg−1, and MQLs were included between 0.07 and 0.88mgkg−1. Due to matrix effect, quantitation was performed by referring to a matrix matched calibration curve, for each cheese typology. This method was also applied to commercial cheese samples, with good results. It appears fast, reliable and suitable for both screening and confirmation of the presence and quantitation of the preservatives in a single, multi-detection analysis.

Highlights

► An RP-HPLC–ESI-MS/MS based method was developed for the multi-determination of seven preservatives in three typologies of cheeses. ► The analytical strategy was evaluated in terms of accuracy, precision, limit of detection, and limit of quantification. ► Our method was applied to commercial cheese samples with good results.

Novel affinity purification of xanthine oxidase from Arthrobacter M3

27 September 2012, 09:32:50
Publication year: 2012
Source:Journal of Chromatography B, Volume 906
Yuran Zhang, Yu Xin, Hailin Yang, Ling Zhang, Xiaole Xia, Yanjun Tong, Yi Chen, Li Ma, Wu Wang
An affinity protocol for purification of xanthine oxidase (XOD) from Arthrobacter M3 was developed. The isolation procedure consisted of only three steps, ammonium sulfate precipitation, affinity extraction to exclude the major impurities, and the final refining procedure with DEAE ion-exchange chromatography for removal of minor contaminants. In this affinity preparation, guanine, an analogue of xanthine, was chosen as the affinity ligand, and was coupled with Sepharose 4B through spacers composed of epichlorohydrin and ethylenediamine. Crude protein has been run through ammonium sulfate precipitation and the affinity column, 99.1% of proteins were removed. After DEAE ion-exchange chromatography, the purity of the refined XOD was 97.5% by Native-PAGE analysis. The activity recovery of purified XOD (36.1%) was almost higher than that of other methods reported. Reducing SDS-PAGE analysis showed that the purified XOD (one band in Native-PAGE analysis) showed two polypeptides with the molecular weights ∼35kDa and ∼100kDa, respectively. The desorption constant K d and the theoretical maximum absorption Q max on the affinity medium were 3.0μg/ml and 2.2mg/g medium in absorption analysis.

Highlights

► A novel affinity protocol for the purification of xanthine oxidase is developed. ► Only three steps successfully purified xanthine oxidase. ► The most important step of this protocol is affinity chromatography. ► The mechanism relies on the affinity interaction between ligand and the enzyme. ► This method has advantage of fewer steps, better recoveries, and higher purity.

Development of a sensitive HPLC method to measure in vitro permeability of E- and Z-isomeric forms of thiosemicarbazones in Caco-2 monolayers

27 September 2012, 09:32:50
Publication year: 2012
Source:Journal of Chromatography B, Volume 906
Zufan Debebe, Sergei Nekhai, Meseret Ashenafi, David B. Lovejoy, Danuta S. Kalinowski, Victor R. Gordeuk, W. Malcolm Byrnes, Des R. Richardson, Pradeep K. Karla
In the current study, we developed a HPLC method to quantitatively measure the permeability of the BpT-based chelators, 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT) and 2-benzoylpyridine 4-allyl-3-thiosemicarbazone (Bp4aT), across human colorectal adenocarcinoma (Caco-2) monolayers as a model of gut absorption. In aqueous solution, Bp4eT and Bp4aT formed inter-convertible Z and E isomers that were resolved by HPLC. Peak area was linear with respect to chelator concentration. Acceptable within-day and between-day precision (<22%) and accuracy (85–115% of true values) were obtained over a range of 1.0–100μM for Bp4eT and 1.5–300μM for Bp4aT. Limits of detection were 0.3μM and 1μM for Bp4eT and Bp4aT, respectively, while corresponding limits of quantification were 1μM and 5μM. Both chelators showed significant ability to chelate iron in THP-1 cells using a calcein-based assay and no apparent cytotoxicity was observed within 24h. Ratios of the apical to basolateral and basolateral to apical transport for Bp4eT were 1.10 and 0.89 at 100μM and 300μM respectively, indicating equal bi-directional movement of the compounds. Similarly, ratios were 0.77 and 0.92 for Bp4aT, respectively. This study demonstrates that Bp4eT and Bp4aT can be efficiently transported through Caco-2 cells and can potentially be formulated for oral delivery.

Highlights

► We developed a sensitive HPLC method for assessment of BpT-E/Z isomers. ► We demonstrated effective iron chelation of BpT-iron chelators that inhibit HIV-1. ► We estimated the permeability profile of BpT-E/Z isomers in Caco2 monolayers. ► We anticipate that BpT chelators could hold promise as orally effective agents.

Identification of the urinary metabolites of glionitrin A in rats using ultra-performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry

27 September 2012, 09:32:50
Publication year: 2012
Source:Journal of Chromatography B, Volume 906
Soo Hyun Lee, Hyun Ok Yang, Hak Cheol Kwon, Byung Hwa Jung
Glionitrin A (GN A) is a new diketopiperazine disulfide with an aromatic nitro group, which is isolated from the coculture of an Aspergillus fumigatus fungal strain and a Sphingomonas bacterial strain. After intravenous administration of GN A in rats, 13 urinary metabolites of GN A were identified using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectroscopy (UPLC–QTOP-MS) analysis in conjunction with data processing programs such as MetaboLynx™ and MassFragnent™. Reduction, nitro-reduction and hydration were the primary metabolic processes affecting GN A in vivo, followed by demethylation or oxidative deamination to alcohol, as well as cysteine, glycine, glucuronide or sulfate conjugation. The metabolite resulting from reduction was found to be a molecule with a dithiol group, and the metabolite made by nitro reduction was found to be an aromatic amine corresponding to GN A. Both of these products may have pharmacological or toxicological activity, which is valuable information in terms of using GN A as a lead compound. In addition, this work showed that UPLC–QTOP-MS analysis coupled with efficient data processing programs is useful for rapid and reliable characterization of GN A metabolites in vivo.

Highlights

► After intravenous administration of GN A in rats, urinary metabolites of GN A were identified. ► Metabolite identification was performed using UPLC–QTOP-MS analysis in conjunction with data processing programs. ► Reduction, nitro-reduction and hydration were the primary metabolic processes affecting GN A in vivo. ► Demethylation, oxidative deamination to alcohol and conjugations were also included in GN A metabolism.

On-line sample concentration and determination of cationic alkaloids in human plasma by micelle to solvent stacking in capillary zone electrophoresis

27 September 2012, 09:32:50
Publication year: 2012
Source:Journal of Chromatography B, Volume 906
Shuaihua Zhang, Ruiyang Ma, Xiumin Yang, Chun Wang, Zhi Wang
A sensitive method for the determination of three cationic alkaloids (berberine, palmatine and jatrorrhizine) from human plasma samples was developed by micelle to solvent stacking (MSS) in capillary zone electrophoresis (CZE). In MSS, the sample preconcentration mainly relies on the reversal in the effective electrophoretic mobility of the analytes at the boundary zone between the sample and CZE background solution (BGS). Under the optimized conditions, the sensitivity enhancement factors achieved in terms of corrected peak area were in the range from 47 to 53 for the alkaloids. The limits of detection (LODs) (S/N=3) for berberine, palmatine and jatrorrhizine were 0.01, 0.01 and 0.02μg/mL, respectively. The intraday (n =6) and interday repeatabilities (n =12) expressed as the relative standard deviations (RSDs) were less than 6.9% in terms of peak height and less than 7.3% in terms of corrected peak area, respectively. The recoveries of the method for the three alkaloids were in the range of 95.9–101.5% with peak height as the quantitative signal, and 92.6–103.6% with corrected peak area as the quantitative signal, respectively. The MSS-CZE method proved to be suitable for the analysis of the alkaloids in human plasma samples.

Highlights

► On-line sample concentration and analysis of some cationic alkaloids in human plasma. ► Micelle to solvent stacking in capillary zone electrophoresis. ► High sensitivity and good recovery.

Generic and rapid determination of veterinary drug residues and other contaminants in raw milk by ultra performance liquid chromatography–tandem mass spectrometry

27 September 2012, 09:32:50
Publication year: 2012
Source:Journal of Chromatography B, Volume 906
Jia Zhan, Xue-jun Yu, Ying-ying Zhong, Zai-ting Zhang, Xiao-mei Cui, Jin-feng Peng, Rui Feng, Xiao-tao Liu, Yan Zhu
A generic, rapid and simple analytical method able to identify 255 veterinary drug residues and other contaminants in raw milk had been developed. The method was based on two-step simple precipitation and ultra performance liquid chromatography coupled with electrospray ionization and tandem mass spectrometry (UPLC–ESI–MS/MS) operating both in positive and negative multiple reaction mode (MRM). For most of the target analytes, the optimized pretreatment processes led to no significant interference on analysis from complicated sample matrix. For quantification, matrix-fortified calibration curves were performed to compensate for the matrix effect and loss in sample preparation. Competent linearity was found for over 90% of target compounds with linear regression coefficients (R) higher than 0.99. Detection limits ranged from 0.05 to 10μg/kg. Average recoveries spiked into raw milk were in the range from 63% to 141% with associated RSD values from 1% to 29% under the selected conditions. The method had been validated for its extraction sensitivity, linearity, recoveries and precision. The results clearly demonstrated the feasibility of the approach proposed. Application of this method, which improved efficiency and coverage of residues, would imply a drastic reduction of both effort and time in routine monitoring programs.

Highlights

► First, the proposed method was generic for a wide range polarity compounds. ► Second, the method, with only two steps, was rapid and straightforward. ► Third, the way to remove water in milk for concentration was novelty. ► Finally, this kind of method is urgently needed in dairy plants.

Aqueous normal phase liquid chromatography coupled with tandem time-of-flight quadrupole mass spectrometry for determination of zanamivir in human serum

27 September 2012, 09:32:50
Publication year: 2012
Source:Journal of Chromatography B, Volume 906
Jing Ge, Fengmao Liu, Eric H. Holmes, Gary K. Ostrander, Qing X. Li
An aqueous normal phase (ANP) liquid chromatography coupled with a hybrid quadrupole time-of-flight mass spectrometry (ANP-LC-micrOTOFQ) method was used for the determination of zanamivir in human serum. Zanamivir was extracted with methanol from protein-precipitated human serum samples and further purified with SCX solid-phase extraction cartridges. Scherzo SM-C18, Agilent Zorbax SB-Aq, Cogent Diamond Hydride, Cogent Bidentate and Luna HILIC columns were compared and optimized for the retention and separation of zanamivir and the Luna HILIC and Diamond Hydride columns exhibited the best retention of zanamivir. The former provided a shorter retention time, a sharper peak and relatively high sensitivity, whereas the latter exhibited a longer retention time and less matrix interference. The analytical range of the calibration curve was between 5 and 1000ng/mL.

Highlights

► Aqueous normal phase chromatography was studied for the separation of zanamivir in human sera. ► Five different columns were compared for the capability to retain zanamivir. ► Effects of mobile phase constituents on the retention time were examined. ► The Diamond Hydride column was the most effective to retain zanamivir and avoid matrix effects.

Development and validation of a rapid capillary zone electrophoresis method for determining charge variants of mAb

27 September 2012, 09:32:50
Publication year: 2012
Source:Journal of Chromatography B, Volume 906
Ying Shi, Zhen Li, Yuanbiao Qiao, Jun Lin
This work aimed to develop a rapid capillary zone electrophoresis (CZE) method to provide abundant purity and identity information of monoclonal antibodies. The CZE running buffer system was optimized to be 20mM acetate–acetic acid (pH 6.0) together with the co-addition of 0.3% polyethylene oxide (PEO) and 2mM triethylenetetramine (TETA), which was further tested with advantages on the peak resolution improvements. The conditioning period was scheduled to 1min for both 0.1M HCl and CZE running buffer to reduce total separation time. Additionally, the applied voltage and effective separation length were optimized at 30kV and 20cm separately. Compared with the method reported by Yan [1], this newly developed method showed a higher resolution in separating the two unknown basic peaks by testing monoclonal antibody sample (mAb1). The further validation results showed that for all five of charge isoform peaks of test mAb1, repeatability, intraday and interday precision had a RSD less than 0.58% for migration time and less than 3.18% for corrected area percent. The correlation coefficients of more than 0.98 for all peaks also demonstrated the good linearity for the method. In addition to the application of distinguishing intact antibody from C-terminal Lys variants, the method also has advantage in separating the Fab, Fc and intact antibody-relevant substances quickly, which facilitated the rough evaluation of papain induced digestion.

Highlights

► We prepared a simple CZE running buffer. ► We developed and optimized a CZE method in dynamically coated capillary. ► We performed fast separation in less than 5min with high resolution.

Simultaneous determination of azelastine and its major metabolite desmethylazelastine in human plasma using high performance liquid chromatography–tandem mass spectrometry

27 September 2012, 09:32:50
Publication year: 2012
Source:Journal of Chromatography B, Volume 906
Wuyi Zha, Linyee Shum
A selective and sensitive high performance liquid chromatography–tandem mass spectrometric method was developed for the analysis of azelastine and its major metabolite, desmethylazelastine, in human plasma. Azelastine-13C, d3 was used as internal standard. Azelastine, desmethylazelastine and the internal standard were extracted by a liquid–liquid extraction method and separation was performed under isocratic chromatographic condition. An abnormal signal loss issue for desmethylazelastine during method development was investigated and resolved. The developed method was precise and reproducible as shown by good intraday assay and interday assay precision (CV%≤12.8%). The calibration curve was linear over a range of 10.0/10.0–1000/200pg/mL for azelastine/desmethylazelastine. The method was successfully applied to a pilot bioequivalence study subsequently.

Highlights

► LC–MS/MS method for simultaneous quantitation of azelastine and desmethylazelastine. ► Low LOQ of 10pg/mL for both compounds were achieved. ► An abnormal signal loss issue for desmethylazelastine was investigated and resolved.

Simultaneous determination of eight corticosteroids in bovine tissues using liquid chromatography–tandem mass spectrometry

27 September 2012, 09:32:50
Publication year: 2012
Source:Journal of Chromatography B, Volume 906
Ádám Tölgyesi, Virender K. Sharma, Szabolcs Fekete, Dóra Lukonics, Jenő Fekete
This paper describes a newly developed method for the simultaneous determination of eight corticosteroid residues in bovine muscle, liver and kidney samples using liquid chromatography–tandem mass spectrometry (LC–MS/MS). The determination of methylprednisone, the main metabolite of methylprednisolone, in bovine tissues using LC–MS/MS is carried out for the first time. The method development demonstrates that the pH is important in optimizing the sample preparation. Tests performed using different solid-phase extraction (SPE) cartridges were enabled to produce conditions for reducing the matrix effects (ion suppression and enhancement) of analysis. Acidic condition and mixed-mode cation exchange SPE columns resulted in the most suitable clean-up for muscle and liver, and also yielded acceptable results for kidney. The enhanced sample clean-up resulted in excellent clear baselines of ion transitions, and therefore, a higher delta electron multiplier voltage (ΔEMV) could be set in the MS/MS detector. The application of 500V of ΔEMV improved the signal responses, however, the noise level did not change, and consequently, the overall sensitivity and analytical limits (limit of detection, limit of quantification) could be enhanced. In the HPLC separation, the recently introduced Kinetex phenyl-hexyl core–shell type column was used that enabled baseline separation for dexamethasone and its β-epimer, betamethasone. Dexamethasone and betamethasone were eluted within 12min and such reduced retention, obtained with core–shell HPLC type column, further enhanced the sensitivity. The method was validated according to the European Union (EU) 2002/657/EC Decision; the studied parameters met the EU standards. The decision limits and limit of detections were calculated in each matrix for all corticosteroids and varied from 0.01 to 13.3μg/kg and from 0.01 to 0.1μg/kg, respectively.

Highlights

► A new LC–MS/MS method is developed for corticosteroids in tissue samples. ► Acidic pH control and mixed-mode SPE cartridges are essential for sample clean-up. ► Corticosteroid epimers can be separated simultaneously using Kinetex HPLC column. ► The enhanced clean-up and LC–MS/MS separation improved the analytical limits. ► The method is successful in analyzing dexamethasone in incurred samples.

Validation of a chiral liquid chromatography–tandem mass spectrometry method for the determination of pantoprazole in dog plasma

27 September 2012, 09:32:50
Publication year: 2012
Source:Journal of Chromatography B, Volume 906
Meixia Chen, Yu Xia, Zhiyu Ma, Liang Li, Dafang Zhong, Xiaoyan Chen
Pantoprazole (PAN), a selective proton pump inhibitor, is used clinically as a racemic mixture for the treatment of acid-related gastrointestinal disorders. To investigate its stereoselective pharmacokinetics, a chiral liquid chromatography–tandem mass spectrometry method was developed and validated to determine the pantoprazole enantiomers in dog plasma. After liquid–liquid extraction, a baseline resolution of enantiomers was achieved on an ovomucoid column using the mobile phase of methanol:acetonitrile:10mM ammonium formate (pH 7) (10.4:2.6:87, v/v/v) at 30°C within 10min. Stable isotopically labeled (+)-d3-pantoprazole and (−)-d3-pantoprazole were used as internal standards. Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode via positive atmospheric pressure chemical ionization. The method was linear in the concentration range of 20.0–10,000ng/mL for each enantiomer using 25μL of dog plasma. The lower limit of quantification (LLOQ) for each enantiomer was 20.0ng/mL. Intra- and inter-day precision ranged from 3.2% to 10.3% for (+)-pantoprazole and 3.7–10.0% for (−)-pantoprazole. Accuracy varied from −1.4% to −0.2% for (+)-pantoprazole and −1.6% to 0.8% for (−)-pantoprazole. The validated method was applied successfully for stereoselective pharmacokinetic studies of racemic pantoprazole.

Highlights

► A chiral LC–MS/MS method was validated to quantify pantoprazole enantiomers. ► Separation was performed on an ovomucoid protein column using MS compatible mobile phases. ► Baseline resolution within 10min leads to a reduction in the overall analysis time.

Rapid resolution liquid chromatography (RRLC) analysis of amino acids using pre-column derivatization

27 September 2012, 09:32:50
Publication year: 2012
Source:Journal of Chromatography B, Volume 906
Xiaoli Zhang, Tong Zhao, Ting Cheng, Xiaoyan Liu, Haixia Zhang
A rapid resolution liquid chromatography (RRLC) method was developed for the simultaneous determination of 23 amino acids in rat serum after pre-column derivatization with 2,4-dinitrofluorobenzene (DNFB). The amino acid derivatives were separated on an Agilent Zorbax Eclipse Plus C18 (4.6mm×50mm, 1.8μm) column at 45°C. Ultraviolet (UV) detection was set at 360nm. Good separation of 23 amino acids was achieved within 10min with a ternary gradient elution of mobile phase at a flow rate of 1.5mLmin−1. Calibration curves were linear over the range from 1 to 500μmolL−1 with coefficients 0.9962 or better for each amino acid. The lower limits of quantification (LLOQ) of all 23 amino acids were 1μmolL−1 with signal-to-noise (S/N) ratio ≥4. Intra- and Inter-day precisions, expressed as relative standard deviation (RSD) percentages, were ranged from 0.32% to 3.09% and 0.67% to 5.82%, respectively. Finally, it was successfully applied to the determination of amino acids in rat serum with recoveries ranged from 90.8% to 106.0% and RSD percentages ranged from 1.78% to 4.68%, respectively. The results showed that the proposed method provided a shorter elution time, better resolution and sharper peak shapes for all amino acids. Compared with the conventional high performance liquid chromatography (HPLC) methods, even some ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS), the established RRLC method was superior performance.

Highlights

► The RRLC method was the first time used for amino acids detection. ► Compared with common HPLC and some UPLC, the RRLC was superior performance. ► The reduced solvent consumption was friendly to environment protection. ► The RRLC based on UV detection was economic and available in common laboratories.

Capillary electrophoresis to quantitate gossypol enantiomers in cotton flower petals and seed

27 September 2012, 09:32:50
Publication year: 2012
Source:Journal of Chromatography B
Sergey Vshivkov, Egor Pshenichnov, Zamira Golubenko, Alik Akhunov, Shadman Namazov, Robert D. Stipanovic
Gossypol is a toxic compound that occurs as a mixture of enantiomers in cotton plant tissues including seed and flower petals. The (-)-enantiomer is more toxic to non-ruminant animals. Efforts to breed cottonseed with a low percentage of (-)-gossypol requires determination of the (+)- to (-)-gossypol ratio in seed and flower petals. We report a method to quantitatively determine the total gossypol and percent of its enantiomers in cotton tissues using high performance capillary electrophoresis (HPCE). The method utilizes a borate buffer at pH 9.3 using a capillary with internal diameter of 50μm, effective length of 24.5cm, 15kV and cassette temperature of 15°C. This method provides high accuracy and reproducible results with a limit of detection of the individual enantiomers of less than 36ng/mL providing base line separation in less than 6minutes.

Highlights

► Gossypol occurs as a mixture of enantiomers in cottonseed. ► (-)-Gossypol is more toxic to non-ruminant animals. ► Plant breeders are developing plants with a low percent of (-)gossypol. ► A capillary electrophoresis method was developed to quantitate the enantiomers. ► Breeders can use this method to select plants with the low (-)gossypol seed trait.

Characterization and identification of alanine to serine sequence variants in an IgG4 monoclonal antibody produced in mammalian cell lines

27 September 2012, 09:32:50
Publication year: 2012
Source:Journal of Chromatography B
Jinmei Fu, Jacob Bongers, Li Tao, Dan Huang, Richard Ludwig, Yunping Huang, Yueming Qian, Jonathan Basch, Joel Goldstein, Ramji Krishnan, Li You, Zheng Jian Li, Michael J. Grace, Reb J. Russell
Low levels of alanine to serine sequence variants were identified in an IgG4 monoclonal antibody by ultra/high performance liquid chromatography and tandem mass spectrometry. The levels of the identified sequence variants A183S and A152S, both in the light chain, have been determined to be 7.8-9.9% and 0.5-0.6%, by extracted ion currents of the tryptic peptides L16 and L14, respectively. The A183S variant was confirmed through tryptic map spiking experiments using synthetic peptide, SDYEK, which incorporated Ser at the position of native Ala in the tryptic peptide L16. Both mutations were also observed by endoproteinase Asp-N peptide mapping. The variant level of A183S was also quantified by LC-UV with detection at 280nm and fluorescence detection of tyrosine residues on the tryptic peptides. The results from LC-MS, UV, and fluorescent detection are in close agreement with each other. The levels of the sequence variants are comparable among the antibody samples manufactured at different scales as well as locations, indicating that the variants’ levels are not affected by manufacture scale or locations. DNA sequencing of the master cell bank revealed the presence of mixed bases at position 183 encoding both wild and mutated populations, whereas bases encoding the minor sequence variant at position152 were not detected. The root cause for A152S mutation is not yet clearly understood at this moment.

Highlights

► Alanine to serine sequence variants were identified in an IgG4 monoclonal antibody by LC/MS/MS. ► The sequence variants were confirmed by use of synthetic peptide. ► DNA sequencing of the mater cell bank revealed one variant was caused by mutation at the DNA level.

Simultaneous determination of morinidazole, its N-oxide, sulfate, and diastereoisomeric N+-glucuronides in human plasma by liquid chromatography–tandem mass spectrometry

27 September 2012, 09:32:50
Publication year: 2012
Source:Journal of Chromatography B
Ruina Gao, Dafang Zhong, Ke Liu, Yu Xia, Rongwei Shi, Hua Li, Xiaoyan Chen
Morinidazole is a new third-generation 5-nitroimidazole antimicrobial drug. To investigate the pharmacokinetic profiles of morinidazole and its major metabolites in humans, a liquid chromatography–tandem mass spectrometry method was developed and validated for simultaneous determination of morinidazole, its N-oxide metabolite (M4-1), a sulfate conjugate (M7), and two diastereoisomeric N +-glucuronides (M8-1 and M8-2) in human plasma. A simple acetonitrile-induced protein precipitation was employed to extract five analytes and internal standard metronidazole from 50 μL human plasma. To avoid the interference from the in-source dissociation of the sulfate and achieve the baseline-separation of diastereoisomeric N +-glucuronides, all the analytes were separated from each other with the mobile phase consisting of 10mM ammonium formate and acetonitrile using gradient elution on a Hydro-RP C18 column (50 mm × 2 mm, 4 μm) with a total run time of 5 min. The API 4000 triple quadrupole mass spectrometer was operated under the multiple reaction-monitoring mode using the electrospray ionization technique. The developed method was linear in the concentration ranges of 10.0ng/mL to 12000ng/mL for morinidazole, 1.00ng/mL to 200ng/mL for M4-1, 2.50ng/mL to 500ng/mL for M7, 3.00ng/mL to 600ng/mL for M8-1, and 10.0ng/mL to 3000ng/mL for M8-2. The intra- and inter-day precisions for each analyte met the accepted value. Results of the stability of morinidazole and its metabolites in human plasma were also presented. The method was successfully applied to the clinical pharmacokinetic studies of morinidazole injection in healthy subjects, patients with moderate hepatic insufficiency, and patients with severe renal insufficiency, respectively.

Highlights

► Simultaneously determine morinidazole and its four metabolites in human plasma. ► Gradient elution was used to obtain the resolution of two N+-glucuronide isomers. ► The potential interference from the in-source dissociation of the conjugates was avoided. ► The method was applied to clinical pharmacokinetic studies of morinidazole injection.

Short-incubation mass spectrometry assay for lysosomal storage disorders in newborn and high-risk population screening

27 September 2012, 09:32:50
Publication year: 2012
Source:Journal of Chromatography B
Thomas P. Mechtler, Thomas F. Metz, Hannes G. Müller, Katharina Ostermann, Rene Ratschmann, Victor R. De Jesus, Bori Shushan, Joseph M. Di Bussolo, Joseph L. Herman, Kurt R. Herkner, David C. Kasper
The interest in early detection strategies for lysosomal storage disorders (LSDs) in newborns and high-risk population has increased in the last years due to the availability of novel treatment strategies coupled with the development of diagnostic techniques. We report the development of a short-incubation mass spectrometry-based protocol that allows the detection of Gaucher, Niemann-Pick A/B, Pompe, Fabry and mucopolysaccharidosis type I disease within four hours including sample preparation from dried blood spots. Optimized sample handling without the need of time-consuming offline preparations, such as liquid-liquid and solid-phase extraction, allows the simultaneous quantification of five lysosomal enzyme activities using a cassette of substrates and deuterated internal standards. Applying incubation times of 3h revealed in intra - day CV% values ranging from 4% to 11% for all five enzyme activities, respectively. In a first clinical evaluation, we tested 825 unaffected newborns and 16 patients with LSDs using a multiplexed, turbulent flow chromatography–ultra high performance liquid chromatography–tandem mass spectrometer assay. All affected patients were identified accurately and could be differentiated from non-affected newborns. In comparison to previously published two-day assays, which included an overnight incubation, this protocol enabled the detection of lysosomal enzyme activities from sample to first result within half a day.

Highlights

► We report a short-incubation mass spectrometry-based protocol for lysosomal enzymes. ► Optimized sample handling and online clean-up allowed a 4h analysis. ► Up to 5 lysosomal enzyme activities are analyzed simultaneously from dried blood spots. ► Method validation was performed under routine clinical laboratory environment.

Determination of five di-(2-ethylhexyl)phthalate metabolites in urine by UPLC-MS/MS, markers of blood transfusion misuse in sports

27 September 2012, 09:32:50
Publication year: 2012
Source:Journal of Chromatography B
Núria Monfort, Rosa Ventura, Georgina Balcells, Jordi Segura
Di-(2-ethylhexyl)phthalate (DEHP) is the most commonly used plasticizer for polyvinyl chloride, which is found in a large variety of products, including most of the bags used for blood storage because of its protective role on erythrocytes survival. DEHP metabolites have been recently proposed as markers of the misuse of blood transfusion in athletes. In this study, a method to quantify the main five DEHP metabolites in urine has been developed: mono-(2-ethylhexyl)phthalate (MEHP), mono-(2-ethyl-5-hydroxyhexyl)phthalate (MEHHP), mono-(2-ethyl-5-oxohexyl)phthalate (MEOHP), mono-(2-ethyl-5-carboxypentyl)phthalate (5cx-MEPP), and mono-(2-carboxymethylhexyl)phthalate (2cx-MMHP). The method involved an enzymatic hydrolysis with β-glucuronidase from Escherichia coli followed by an acidic extraction with ethyl acetate. The hydrolyzed extracts were analysed by ultraperformance liquid chromatography tandem mass spectrometry. Isotope labelled MEHP, MEOHP and 5cx-MEPP were used as internal standards. Analysis of all the metabolites was achieved in a total run time of 10min, using a C18 column and a mobile phase containing deionized water and acetonitrile with formic acid, with gradient elution at a flow-rate of 0.6mLmin−1. Detection of the compounds was performed by multiple reaction monitoring, using electrospray ionization in positive and negative ion modes. The method was validated for quantitative purposes. Extraction recoveries were greater than 90% and the limits of quantitation ranged from 1.2 to 2.6ngmL−1. Intra-day precisions were better than 8% for all metabolites while inter-assay precisions were better than 12%. Concentrations of DEHP metabolites were measured in a control group (n=30, subjects reflecting the common environmental DEHP exposure), and in sportsmen (n=464), to evaluate population distribution exposure to DEHP. Additionally, threshold concentrations indicating outliers of common exposure for DEHP metabolites are proposed.

Highlights

► Di-(2-ethylhexyl)phthalate (DEHP) is the most commonly used plasticizer for PVC ► DEHP metabolites have been proposed as markers of the misuse of blood transfusion ► A method to quantify five DEHP metabolites has been developed and validated ► Cutoff concentrations were proposed to determine suspicions of a possible transfusion ► The method is proposed as screening test for transfusion detection in doping control


The next generation of inverted microscope systems has arrived


Olympus has released the innovative new IX3 series of inverted research microscope systems for effortless, intuitive live cell imaging and clinical analysis. This includes the fully automated IX83 for high-end research applications, the flexible IX73, which can be configured in manual, semi-motorised or motorised modes, and the easy-to-use IX53 with fluorescent capabilities, which is optimised for the routine examination of tissue samples. Built using worldwide customer feedback and designed to meet the needs of a wide range of users, the new systems offer exceptional ease-of-use and unprecedented optical flexibility via a new, customisable light path. New components can be easily slid into the light path using a series of swappable decks. This opens up many new avenues for exploration, allowing researchers to follow their imaginations. The new systems also utilise the latest Olympus innovations in frame design, optics and software, providing exceptional stability, optical quality, accuracy and reliability.
The new IX3 inverted microscope systems are optimised for live cell imaging. In particular, the IX73 and IX83 are built using a new swappable deck design, which allows optical modules to be easily and rapidly slipped in and out as needed, including magnification changers, filter turrets, a right side port and more. This means that the systems are truly ‘open access’ and can be fully customised to meet the requirements of a wide range of applications, even allowing the utilisation of user-designed custom modules to provide the ultimate level of flexibility. This design also allows the systems to grow alongside the evolving demands of your life science research projects. This is especially true of the IX73, which is highly configurable and capable of integrating with a range of computer-coded or motorised components.
The new frame design of the IX3 systems provides an excellent level of mechanical and thermal stability, and includes an ultrasonic motorised stage for quiet, smooth and precise movement. Olympus UIS2 optics deliver sharp, bright images, while the proprietary Olympus fly-eye fluorescence illuminator generates even, vivid illumination across the specimen. This provides a much wider field of view than previously possible, with even illumination that fills the detectors of most cameras, even those with larger chips. IX3 systems are also compatible with the Olympus Real-Time Controller, offering high-precision imaging via microsecond synchronisation for advanced studies using high-speed light sources and triggered cameras.
Optimised for ease-of-use, the IX3 systems also help streamline complex imaging and measurement tasks. The systems integrate seamlessly with the powerful Olympus cellSens imaging software to allow advanced analysis at the click of a button, while the IX83 also offers a touch-panel interface that can be located in the most ergonomic position for each user, providing simple, quick and comfortable operation. With live cell imaging in mind, the IX83 is also compatible with the new Olympus ZDC Z-Drift Compensation system, which automatically ensures that every image you capture is in perfect focus. In particular, the ZDC can work independently of software control, and uniquely combines a one-shot and continuous mode in the same unit, ensuring your system can be easily optimised for a wide range of different applications. 
Built to provide the highest-level of quality, flexibility and reliability, the IX3 series of microscope systems will meet all your research and examination needs, now and in the future.

Wednesday, 26 September 2012

Just Published: Journal of Chromatography A


A new issue of this journal has just been published. To see abstracts of the papers it contains (with links through to the full papers) click here:
Journal of Chromatography A
Selected papers from the latest issue:

Graphene-supported zinc oxide solid-phase microextraction coating with enhanced selectivity and sensitivity for the determination of sulfur volatiles in Allium species

26 September 2012, 10:35:32
Publication year: 2012
Source:Journal of Chromatography A, Volume 1260
Suling Zhang, Zhuo Du, Gongke Li
A graphene-supported zinc oxide (ZnO) solid-phase microextraction (SPME) fiber was prepared via a sol–gel approach. Graphite oxide (GO), with rich oxygen-containing groups, was selected as the starting material to anchor ZnO on its nucleation center. After being deoxidized by hydrazine, the Zn(OH)2/GO coating was dehydrated at high temperature to give the ZnO/graphene coating. Sol–gel technology could efficiently incorporate ZnO/graphene composites into the sol–gel network and provided strong chemical bonding between sol–gel polymeric SPME coating and silica fiber surface, which enhanced the durability of the fiber and allowed more than 200 replicate extractions. Results indicated that pure ZnO coated fiber did not show adsorption selectivity toward sulfur compounds, which might because the ZnO nanoparticles were enwrapped in the sol–gel network, and the strong coordination action between Zn ion and S ion was therefore blocked. The incorporation of graphene into ZnO based sol–gel network greatly enlarged the BET surface area from 1.2m2/g to 169.4m2/g and further increased the adsorption sites. Combining the superior properties of extraordinary surface area of graphene and the strong coordination action of ZnO to sulfur compounds, the ZnO/graphene SPME fiber showed much higher adsorption affinity to 1-octanethiol (enrichment factor, EF, 1087) than other aliphatic compounds without sulfur-containing groups (EFs<200). Also, it showed higher extraction selectivity and sensitivity toward sulfur compounds than commercial polydimethylsiloxane (PDMS) and polydimethylsiloxane/divinylbenzene (PDMS/DVB) SPME fibers. Several most abundant sulfur volatiles in Chinese chive and garlic sprout were analyzed using the ZnO/graphene SPME fiber in combination with gas chromatography–mass spectrometry (GC–MS). Their limits of detection were 0.1–0.7μg/L. The relative standard deviation (RSD) using one fiber ranged from 3.6% to 9.1%. The fiber-to-fiber reproducibility for three parallel prepared fibers was 4.8–10.8%. The contents were in the range of 1.0–46.4μg/g with recoveries of 80.1–91.6% for four main sulfides in Chinese chive and 17.1–122.6μg/g with recoveries of 73.2–80.6% for three main sulfides in garlic sprout.

Highlights

► A graphene-supported zinc oxide SPME fiber via a sol–gel approach was originally developed. ► The selectivity and sensitivity of ZnO/graphene SPME coating to sulfur compounds were excellent. ► The ZnO/graphene SPME was successfully applied to determine sulfur volatiles in complex samples.

Ultra trace determination of fluorobenzoic acids in tap and reservoir water using solid-phase extraction and gas chromatography–mass spectrometry

26 September 2012, 10:35:32
Publication year: 2012
Source:Journal of Chromatography A, Volume 1260
Karsten Müller, Andreas Seubert
A method for the ultra trace analysis of 21 fluorobenzoic acids (FBAs) via GC–MS based on solid-phase extraction (SPE) and derivatization with BF3·MeOH is described. All fluorobenzoic acids were enriched and determined simultaneously. Solid-phase extraction on hydrophilic–lipophilic-balanced reversed-phase cartridges containing a poly(divinylbenzene-co-N-vinylpyrrolidone) polymer allowed a 250-fold enrichment of the acids if 100mL sample volume is used with extraction efficiencies between 71% and 94%. The method enables the determination of fluorobenzoic acid methyl esters (FBAMEs) down to the range of 6–44ngL−1 combined with a fast and easy sample-preparation (pH-adjusting prior to SPE and derivatization within 24h at 64°C directly in the vial). It uses low amounts of chemicals and is adaptable to larger and smaller sample volumes. Simultaneous extraction and determination of 21 fluorinated aromatic acids in reservoir samples with high salinity confirmed the applicability and reproducibility of the method.

Highlights

► New method for the derivatization of fluorobenzoic acids using BF3·MeOH. ► New SPE method using a material never used before for this application. ► Quantitative extraction of all commercially available 23 FBAs. ► Determination of all 23 fluorobenzoic acid methyl esters in the ngL−1 range. ► Capability of the method for highly saline reservoir water samples is shown.

Stir bar sorptive extraction approaches with a home-made portable electric stirrer for the analysis of polycyclic aromatic hydrocarbon compounds in environmental water

26 September 2012, 10:35:32
Publication year: 2012
Source:Journal of Chromatography A, Volume 1260
Xiangju Mao, Bin Hu, Man He, Wenying Fan
In this study, novel off/on-site stir bar sorptive extraction (SBSE) approaches with a home-made portable electric stirrer have been developed for the analysis of polycyclic aromatic hydrocarbon compounds (PAHs). In these approaches, a miniature battery-operated electric stirrer was employed to provide agitation of sample solutions instead of the commonly used large size magnetic stirrer powered by alternating current in conventional SBSE process, which could extend the SBSE technique from the conventional off-site analysis to the on-site sampling. The applicability of the designed off/on-site SBSE sampling approaches was evaluated by polydimethylsiloxane (PDMS) coating SBSE-high performance liquid chromatography-fluorescence detection (HPLC-FLD) analysis of six target PAHs in environmental water. The home-made portable electric stirrer is simple, easy-to-operate, user friendly, low cost, easy-to-be-commercialized, and can be processed in direct immersion SBSE, headspace sorptive extraction (HSSE) and continuous flow (CF)-SBSE modes. Since the stir bar was fixed onto the portable device by magnetic force, it is very convenient to install, remove and replace the stir bar, and the coating friction loss which occurred frequently in conventional SBSE process could be avoided. The parameters affecting the extraction of six target PAHs by the home-made portable SBSE sampling device with different sampling modes were studied. Under the optimum extraction conditions, good linearity was obtained by all of three SBSE extraction modes with correlation coefficient (R) higher than 0.9971. The limits of detection (LODs, S/N=3) were 0.05–3.41ngL−1 for direct immersion SBSE, 0.03–2.23ngL−1 for HSSE and 0.09–3.75ngL−1 for CF-SBSE, respectively. The proposed portable PDMS-SBSE-HPLC-FLD method was applied for the analysis of six target PAHs in East Lake water, and the analytical results obtained by on-site SBSE sampling were in good agreement with that obtained by off-site SBSE sampling. The accuracy of the developed method was evaluated by recovery test and the recoveries for the spiked sample were found to be in the range of 87.1–122.8% for off-site CF-SBSE, 88.8–114.3% for on-site sampling, and 87.7–123.6% for off-site SBSE, respectively. The developed method is one of the most sensitive methods for PAHs determination and the home-designed SBSE system is feasible for the field sampling.

Highlights

► A portable stir bar sorptive extraction system was designed for on-site sampling. ► It can be used for off/on-site sampling with different extraction modes. ► The portable SBSE system was applied for HPLC analyzing six PAHs in lake water. ► The portable SBSE system shows a great application potential in field sampling.

Simultaneous determination of 10 β2-agonists in swine urine using liquid chromatography–tandem mass spectrometry and multi-walled carbon nanotubes as a reversed dispersive solid phase extraction sorbent

26 September 2012, 10:35:32
Publication year: 2012
Source:Journal of Chromatography A, Volume 1260
Xiang-Dang Du, Yin-Liang Wu, Hong-Ju Yang, Ting Yang
A simple and inexpensive pretreatment procedure was developed for 10 β2-agonists (clenbuterol, ractopamine, salbutamol, bambuterol, penbuterol, tulobuterol, clorprenaline, mabuterol, cimaterol and terbutaline) in swine urine using dispersive solid phase extraction (dSPE) with multi-walled carbon nanotubes (MWCNTs). The sample was analysed after purification by ultra high performance liquid chromatography–positive electrospray ionisation tandem mass spectrometry (UHPLC-ESI–MS/MS). The pH value of the swine urine, extraction time, type and amount of MWCNTs and type of eluent were optimised to increase the sample throughput and sensitivity. The samples were quantified using clenbuterol-D9, ractopamine-D6 and salbutamol-D3 as internal standards. The recoveries of the target compounds from swine urine samples at pH 10.0 were most efficient when using 20mg of MWCNTs with a 30–50nm outer diameter and a length of 10–30μm, while a mixture of water/methanol (90:10, v/v) with 0.5% formic acid was shown to be the most suitable solvent for desorbing the compounds from the MWCNTs. The proposed method was validated according to the European Commission Decision 2002/657/EC, which determines linearity, specificity, decision limit (CCα), detection capability (CCβ), recovery, precision and stability.

Highlights

► A simple and cheap method was developed for β2-agonists using dSPE with MWCNTs. ► The pH, extraction time, the type and amount of MWCNTs were optimised. ► The proposed method was validated according to 2002/657/EC. ► The whole method was fast, reliable, convenient and sensitive.

Investigation of protocols to extraction and quantification of folates in vegetables matrices split into liquor and fiber fraction using factorial design

26 September 2012, 10:35:32
Publication year: 2012
Source:Journal of Chromatography A, Volume 1260
Emmanuela Prado de Paiva, Clayton Anderson de Azevedo Filho, Sabrina Gomes Ferreira, Tânia Lucia Montenegro Stamford, Jose Almiro da Paixão
The main protocols of extraction were investigated for the six folate forms in vegetable matrices, treated in two fractions, liquor and fiber. In a pilot study, it was used ammonium acetate added of 2-mercaptoetanol and ascorbic acid as extraction solution. The condition of use of protease and folate conjugase was evaluated, besides alternative treatments without enzyme use. Based on the results of this stage, it was built the factorial design 24, with three replications at the central point, using the following variables: temperature, time for reaction, molar concentration of the extraction solution and ratio sample/solution as independent variables and dependent variable, the amount of each folate form extracted as well as spectral and chromatographic parameters. In the pilot study it was verified that the enzyme use can cause an increase in the variability of the folate content, which enabled to build the factorial design without the enzyme use. The binomial time and temperature showed greatest impact on the extraction profile, besides high concentrations of ammonium acetate resulting in bifurcation of some peaks. 5-Methyltetrahydrofolate was extracted primordially in the liquor fraction, indicating that this treatment on the matrix provoked suitable extraction condition to this folate.

Highlights

► Statistic factorial design confirms the binomial impact of time and temperature. ► Low temperature and short time are indicated as the best extraction conditions. ► 5-MTHF was suitability extracted from liquor fraction of all vegetable matrices.

Planar solid phase extraction clean-up for pesticide residue analysis in tea by liquid chromatography–mass spectrometry

26 September 2012, 10:35:32
Publication year: 2012
Source:Journal of Chromatography A, Volume 1260
Claudia Oellig, Wolfgang Schwack
Efficient clean-up is indispensable for preventing matrix effects in multi-residue analysis of pesticides in food by liquid and gas chromatography (LC and GC) coupled to mass spectrometry (MS). High-throughput planar solid phase extraction (HTpSPE) was recently introduced as a new clean-up concept in residue analysis of pesticides in fruit and vegetables (C. Oellig, W. Schwack, 2011 [45]). Thin-layer chromatography (TLC) was used to completely separate pesticides from matrix compounds and to focus them into a sharp zone, followed by extraction of the target zone by the TLC–MS interface. As rather challenging matrices, tea samples were chosen in this study. Besides chlorophylls and polyphenols, high amount of caffeine is co-extracted resulting in strong matrix effects both in LC–MS and GC–MS. The former HTpSPE procedure was adapted to initial extracts of green and black tea resulting in colorless extracts nearly free of matrix effects and interferences, as shown for seven chemically representative pesticides (acetamiprid, penconazole, azoxystrobin, chlorpyrifos, pirimicarb, fenarimol, and mepanipyrim). LC–MS/MS calibration curves obtained in the range of 0.002–0.5mg/kg from matrix-matched standards and solvent standards were nearly identical and demonstrated the effectiveness of clean-up by HTpSPE. Mean recoveries determined by LC–MS/MS against solvent standards at spiking levels of 0.01 and 0.1mg/kg ranged between 72 and 114% with relative standard deviations (RSDs) of 0.7–4.7% (n =4), while LC–MS measurements of tea samples spiked at 1mg/kg provided recoveries of 81–104% with RSDs of 1.2–4.9% (n =6). Using LC–MS/MS, the method showed high sensitivity with signal-to-noise ratios >10 for concentrations below 0.002mg/kg. HTpSPE of one sample was done in a few minutes, while numerous samples were cleaned in parallel at minimal costs with very low sample and solvent consumption.

Highlights

► A new clean-up concept for pesticide residue analysis in tea by LC–MS is introduced. ► Planar solid phase extraction was proven to be the highly efficient clean-up method. ► Nearly matrix-free tea extracts generally avoided matrix effects in LC–MS(/MS). ► Applying pure solvent standards, method validation showed impressive results.

Investigation and optimization of particle dimensions for needle trap device as an exhaustive active sampler

26 September 2012, 10:35:32
Publication year: 2012
Source:Journal of Chromatography A, Volume 1260
Weiqiang Zhan, Janusz Pawliszyn
Various needle trap devices (NTDs) with different designs have been developed during the past decade. A theoretical model on the fundamentals of the NTD was recently proposed, which employed the theory of frontal (gas–solid) chromatography to describe the sampling process. In the current work, different types of sorbent particles with different dimensions were packed into the needle as the adsorbent. The effects of particle dimensions, which would affect the packing density and consequently affect the capacity, the extraction efficiency and desorption efficiency of the NTD were experimentally investigated and the proposed theory was validated. The results demonstrated that NTDs packed with small particles possess higher extraction capacity and efficiency but much higher resistances to flow as well. The higher resistance did not necessarily result in poor desorption efficiency. The observed relationships among those physical parameters provide valuable guidance on how to design a NTD with high performance for future applications.

Highlights

► Frontal chromatography process is experimentally validated to describe the sampling process of a needle trap device. ► The trapping efficiency and desorption efficiency of a needle trap device in regard to particle dimensions are investigated and optimized. ► Effects of particles on the extraction of toluene, ethylbenzene and o-xylene are presented.

Process for purification of monoclonal antibody expressed in transgenic Lemna plant extract using dextran-coated charcoal and hexamer peptide affinity resin

26 September 2012, 10:35:32
Publication year: 2012
Source:Journal of Chromatography A, Volume 1260
Amith D. Naik, Stefano Menegatti, Hannah R. Reese, Patrick V. Gurgel, Ruben G. Carbonell
The production of therapeutic proteins using transgenic plants offers several advantages, including low production cost, absence of human pathogens, presence of glycosylation mechanisms, and the ability to fold complex therapeutic proteins into their proper conformation. However, impurities such as phenolic compounds and pigments encountered during purification are quite different from those faced during purification from mammalian cell culture supernatants. This paper deals with the development of a pretreatment and affinity separation process for the purification of a monoclonal antibody from transgenic Lemna plant extract. A pretreatment step is described using dextran-coated charcoal for the removal of pigments and phenolic compounds without reducing the antibody concentration. Then, the peptide affinity ligand HWRGWV coupled to a commercial polymethacrylate resin is used for the capture and purification of MAb from the pretreated plant extract. The final yield and purity of the MAb obtained were 90% and 96% respectively. The performance of the hexamer peptide resin after the pretreatment step was found to be similar to that obtained with a commercial Protein A resin.

Highlights

► This paper deals with the purification of a monoclonal IgG from a plant extract. ► We used a two-step approach, with a charcoal treatment and affinity chromatography step. ► The final product shows high purity and recovery, competitive to commercial products.

Ultra-high performance liquid chromatography coupled to high resolution Orbitrap mass spectrometry for metabolomic profiling of the endogenous phytohormonal status of the tomato plant

26 September 2012, 10:35:32
Publication year: 2012
Source:Journal of Chromatography A, Volume 1260
Lieven Van Meulebroek, Julie Vanden Bussche, Kathy Steppe, Lynn Vanhaecke
Phytohormones are key signalling biomolecules and are of particular interest because of their regulating role in numerous physiological and developmental plant processes. Since the plant response to a given stimulus results amongst others from the complex interaction between phytohormones, there is a mounting interest for multiple phytohormone analysis. Therefore, with the primary aim of profiling the hormonal status of the tomato plant, a generic extraction protocol and an U-HPLC–Orbitrap-MS analysis were developed and validated for both tomato fruit and leaf tissue. To this end, eight phytohormones were considered, i.e. gibberellic acid, indol-3-acetic acid, abscisic acid, jasmonic acid, salicylic acid, zeatin, N6-benzyladenine and epibrassinolide, representing the major hormonal classes. The sample pre-treatment involved liquid extraction with a buffer of methanol, ultrapure water and formic acid (75:20:5, v/v/v), after which the extract was purified by means of an Amicon® Ultra centrifugal unit. Subsequently, analytes were chromatographically separated on a sub-2μm particles Nucleodur Gravity C18 column and detected by an Exactive™ high-resolution mass spectrometer. Validation of the analytical method demonstrated that linearity (≥0.99), precision (CV≤15%) and mean corrected recovery (between 80% and 110%) performed well for the majority of the eight targeted phytohormones. In addition, the generic nature of the extraction protocol and the full scan approach of the Orbitrap mass spectrometer allowed metabolomic profiling of the hormonal status of the tomato plant.

Highlights

► Development of a generic extraction for phytohormones from tomato plant tissue. ► Development of a full scan U-HPLC–Orbitrap-MS method for phytohormone analysis. ► Validation of the developed methods for both tomato leaf and fruit tissue. ► Demonstration of the metabolomic character of the developed methods.

Improved chemical stabilities for end-capped high performance liquid chromatography stationary phases based on poly(methyloctadecylsiloxane) thermally immobilized onto metalized silicas

26 September 2012, 10:35:32
Publication year: 2012
Source:Journal of Chromatography A, Volume 1260
Carla G.A. da Silva, Carol H. Collins
Endcapped stationary phases were prepared after thermal immobilization of poly(methyloctadecylsiloxane) (PMODS) onto zirconized and titanized silica supports. These new stationary phases have lower densities of residual hydroxyl groups, according to infrared spectroscopy and 29Si CP-MAS NMR and as shown by the symmetrical peaks of basic compounds from the Tanaka, Engelhardt and SRM 870 test mixtures. Stability tests for the endcapped stationary phases, measured using severe alkaline conditions (70:30 (v/v) methanol:0.05mol/L K2CO3/KHCO3, pH 10, 50°C), revealed that the stabilities of these phases are greater than the stabilities of similar nonendcapped phases. The stationary phases showed good performance for the separation of basic pharmaceuticals.

Highlights

► HPLC support prepared by metallization of silica and thermal immobilization of PMODS onto the support. ► Endcapping reaction with hexamethyldisilazane (HMDS) and trimethylchlorosilane (TMCS). ► Evaluation with several standardized test mixtures. ► Improvement of the chemical stability in severe alkaline conditions. ► Potential application for separation of basic pharmaceuticals.

Suitability of commercial hydrophobic interaction sorbents for temperature-controlled protein liquid chromatography under low salt conditions

26 September 2012, 10:35:32
Publication year: 2012
Source:Journal of Chromatography A, Volume 1260
Tobias K.H. Müller, Matthias Franzreb
The effect of temperature in the range from 10°C to 40°C and comparatively low ammonium sulfate (AS) concentrations of up to 0.5M on the adsorption of bovine serum albumin (BSA) on four different commercially available sepharose-based stationary phases was investigated. The determined isotherms were fitted by the Langmuir equation, and thermodynamic values were calculated by van’t Hoff analysis. The adsorption of BSA onto the chromatographic resin Butyl Sepharose 4FF showed the strongest temperature influence; however, protein unfolding effects occurred when characterizing this system by dynamic column experiments, with an unfolded BSA fraction strongly attached to the sorbent. The percentage of the unfolding fraction was determined for different operating conditions and found to increase with the concentration of the cosmotropic salt, but even stronger with increasing temperature. Temperature-induced cyclic adsorption and desorption experiments were carried out to investigate the long-term performance of Butyl Sepharose 4FF by applying purely temperature-controlled regeneration. Over a period of five cycles, the working capacity remained stable, but BSA also started to accumulate on the column due to incomplete regeneration. Finally, the possibility to fractionate different proteins with a single temperature shift was shown by the complete separation of lysozyme and BSA. The results presented indicate that temperature-induced binding and elution may offer a possibility to shift the operation conditions of HIC resins toward reduced salt concentrations, thus saving chemicals and facilitating salt removal in further downstream processing stages.

Highlights

► Temperature-dependent adsorption behavior of model proteins on HIC gels. ► Temperature as advantageous parameter regarding purification applications using HIC. ► Non-isothermal HIC resin regeneration. ► Temperature-mediated protein fractionation. ► Protein unfolding in dependence of ionic strength, temperature and pulse injections.

Size-exclusion-chromatography separation of randomly branched polymers with tetrafunctional branch points and local dispersity

26 September 2012, 10:35:32
Publication year: 2012
Source:Journal of Chromatography A, Volume 1260
Miloš Netopilík
The SEC separation of a randomly branched polymer, in particular local dispersity due to branching, are theoretically examined. A model of the SEC separation of randomly branched polymer with tetrafunctional branch points enabling the estimation of local dispersity was developed. Measurable quantities (branching indices) were compared with real data. Local dispersity due to branching is demonstrated to depend on elution volume and degree of branching and in the area of the beginning of the elution curve it can reach non-negligible values.

Highlights

► SEC analysis of randomly branched polymer was modeled. ► Results were compared with experiment. ► Local dispersity in the frontal area of the elution curve was found.

Application of Doehlert uniform shell designs for selecting optimal amounts of internal standards in the analysis of prostaglandins and leukotrienes by liquid chromatography–tandem mass spectrometry

26 September 2012, 10:35:32
Publication year: 2012
Source:Journal of Chromatography A, Volume 1260
Pedro Araujo, Steve Janagap, Elisabeth Holen
A protocol for the analysis of multiple prostaglandins and leukotrienes in cell culture media by using multiple internal standards was validated. A two-factor Doehlert design was used to determine the behaviour of the relationship analyte/internal standard (namely: PGE2/PGE2-d 4, PGE3/PGE2-d 4, LTB4/LTB4-d 4 and LTB5/LTB4-d 4) and to select the optimal amounts of deuterated internal standards for quantifying simultaneously the prostaglandins and leukotrienes in cell culture media by LC–MS/MS. The selection of optimal amounts of internal standards was based on mathematical models that allow visualizing concentration regions where the response factors remain constant over a wide range of analytical concentrations. The linearity of the calibration curves for each analyte at the optimal levels suggested by the mathematical models was statistically confirmed by means of the ratio lack-of-fit to pure error. The validated protocol was successfully applied in the simultaneous quantification of pro- and anti-inflammatory eicosanoids in stimulated cod head kidney cell culture media. The two-factor Doehlert design has permitted to estimate the experimental response as a function of six variables (PGE2, PGE3, LTB4, LTB5, PGE2-d 4 and LTB4-d 4) which represents a substantial reduction of resources, time and experiments of approximately 84% (7×3 experiments) when compared with the full six-factor Doehlert design (43×3 experiments).

Highlights

► Rational design for selecting optimal amounts of multiple internal standards. ► Study of six variables with a Doehlert design intended for two variables. ► 84% reduction in resources compared to a six-factor Doehlert design.

Determination of sedatives and adrenergic blockers in blood meal using accelerated solvent extraction and Orbitrap mass spectrometry

26 September 2012, 10:35:32
Publication year: 2012
Source:Journal of Chromatography A, Volume 1260
Jeong-Heui Choi, Marc Lamshöft, Sebastian Zühlke, Ki Hun Park, Jae-Han Shim, Michael Spiteller
The detection of veterinary drugs in blood meal is needed since it is used as an environment-friendly agricultural material despite its origination from animal blood. A method using accelerated solvent extraction and liquid chromatographic linear ion trap quadrupole Orbitrap mass spectrometry was developed to determine sedatives and adrenergic blockers in blood meal. The determination method was established following optimizations of accelerated solvent extraction, dispersive solid-phase extraction and high resolution mass spectrometric detection. Linearity, sensitivity, accuracy, repeatability and reproducibility of the method were fully validated. The method was applied to commercial blood meal products.

Highlights

► Combination of ASE and LC–Orbitrap MS for sedatives and α-, β-blockers in blood meal. ► Dispersive solid-phase extraction as an efficient cleanup method. ► Good results of the sensitivity, linearity, accuracy and precision. ► Finding a metabolite of xylazine via the high resolution MS detection.